The mechanism by which metalloenzyme cytochrome c oxidase operates will be studied. Cytochrome oxidase sustains electron flow through a cascade of respiratory enzymes in mitochondrion by catalyzing reduction of oxygen into water at the binuclear heme a3/CuB active site. Cytochrome oxidase harnesses the energy released in this process and utilizes it for transmembrane pumping of protons thus directly contributing to the buildup of chemiosmotic proton gradient. Cytochrome oxidase recently joined a new class of metalloradical enzymes.The unique histidine-tyrosine cross-linked site is oxidized to a radical. Structural position of the radical site as a ligand to CuB strongly suggests its role in both the dioxygen reduction and proton pumping. Due to the structure of the active site, radical species have neither been directly observed nor characterized using traditional spectroscopic techniques. We intend to overcome this limitation by using two new and unique instruments, which bridge three spectroscopic domains - electronic absorption, infra-red vibrational and electron paramagnetic - thus dramatically improving resolution and sensitivity. Our recent results show that aromatic radicals can be detected and characterized by their UV absorption even in large proteins. Using new methodology we will investigate key models radicals, namely tyrosine, tryptophan, histidine and the model for histidine-tyrosine site, with the emphasis on their UV absorption and vibrational properties. The results will be applied to detection of elusive radicals in cytochrome oxidase. Protonation states of radical sites at critical stages of the catalytic cycle will be particularly investigated because of the central role protons play in energy conservation by cytochrome oxidase. The techniques described in this proposal and anticipated results will be directly applicable for a broad range of medically relevant enzymes. Application of UV spectroscopy for radical detection will provide a simple and accessible tool for an early detection and kinetic studies of transient radicals in the rapidly growing family of metalloradical enzymes.